This invention relates to an isolated luminal binding protein promoter sequence and methods for its use.
Molecular Chaperone Proteins
Luminal binding proteins (BiP) have been identified as a type of molecular chaperone localized within the endoplasmic reticulum (ER) and nuclear envelope of eukaryotic cells. BiP is a member of the heat-shock protein 70 (HSP70) family of proteins (Haas, Experimentia 50:1012-1020, 1994). BiP has been found to assist in the co-translational translocation of newly synthesized polypeptides across the ER membrane in yeast (Vogel et al., J. Cell Biol. 110:1885-1895, 1990; and Nguyen et al., Proc. Natl. Acad Sci. USA 88:1565-1569, 1991). BiP remains associated with polypeptides until they attain their properly folded conformation and/or subunit assembly. For polypeptides that are unable to attain their mature conformation due to misfolding (Schmitz et al., EMBO J. 14:1091-1098, 1995) or lack of a subunit component (Knittler et al., Proc. Natl. Acad Sci. USA 92:1764-1768, 1995), BiP remains associated with the polypeptide until the polypeptide is degraded.
In angiosperms, the expression of BiP is subject to developmental, hormonal, stress-induced, and diurnal regulation (Denecke et al., Plant Cell 3:1025-1035, 1991; Jones et al., Plant Physiol. 97:456-459, 1991; Anderson et al., Plant Physiol. 104:1359-1370, 1994; Kalinski et al., Planta 195:611-621, 1995; and Figueiredo et al., Braz. J. Plant Physiol. 9:103-110, 1997). BiP associates with the bean storage protein phaseolin (D""Amico et al., Plant J. 2:443-455, 1992; Pedrazzini et al., Plant J. 5:103-110, 1994) and with rice prolamines (Li et al., Science 262:1054-1056, 1993). High levels of BiP expression are associated with the accumulation of protein intermediates that are unable to attain their proper folded conformation because of mutations such as those seen in the maize zein regulatory mutants xe2x80x9cfloury-2,xe2x80x9d xe2x80x9cdefective endosperm-B30,xe2x80x9d and xe2x80x9cmucronatexe2x80x9d (Boston et al., Plant Cell 3:497-505, 1991; Fontes et al., Plant Cell 3:483-496, 1991).
Treatment with tunicamycin, which inhibits N-linked glycosylation and proper protein folding, also results in increased levels of BiP expression (Denecke et al., Plant Cell 3:1025-1035, 1991; and D""Amico et al., Plant J. 2:443-455, 1992). However, the increased expression resulting from unfolded proteins and from increased levels of secretory protein traffic may be mediated through different signals (Pahl et al., EMBO J. 14:2580-2588, 1995).
The invention provides a Douglas-fir (Pseudotsuga menziesii) luminal binding protein promoter (PmBiPPro1; SEQ ID NO: 31). Expression of the PmBiP protein (SEQ IDNO: 36) is shown herein to be developmentally-regulated and inducible by environmental changes. The promoter (PmBiPPro1; SEQ ID NO: 31), fragments thereof, and variants thereof are useful for expressing heterologous proteins either transiently in host cells or transgenically in stably transformed cells and plants.
One aspect of the invention provides the PmBiP promoter (SEQ ID NO: 31), fragments/deletions of the PmBiP promoter (SEQ ID NO: 31) and variants thereof. The variant promoters are characterized by their retention of at least 50% sequence identity with the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31, respectively), or by their retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31). In each case these promoters, at a minimum, retain promoter activity. In some cases these promoters retain native PmBiP promoter activity.
It is also contemplated that promoters such as the CaMV35S promoter may be altered through the introduction of sequences found in the PmBiP promoter (SEQ ID NO: 31). The resulting promoter also will be characterized by its retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 16, 17, 18, and 31).
Another aspect of the invention provides vectors containing the above-described promoters and variants thereof. The vectors can be transformed into host cells. If the host cell is a plant cell, the transformed host cell can give rise to a transgenic plant.
The invention also provides transgenes. These transgenes include one of the above-described promoter sequences operably linked to one or more open reading frames (ORFs). The transgenes can be cloned into vectors and subsequently used to transform host cells such as bacterial, insect, mammalian, fungal, yeast, or plant cells.
Accordingly, the invention provides transgenic plants such as maize, wheat, rice, millet, tobacco, sorghum, rye, barley, brassica, sunflower, seaweeds, lemna, oat, soybean, cotton, legumes, rape/canola, alfalfa, flax, sunflower, safflower, brassica, cotton, flax, peanut, and clover; lettuce, tomato, cucurbits, cassava, potato, carrot, radish, pea, lentil, cabbage, cauliflower, broccoli, Brussel sprouts, peppers and other vegetables; citrus, apples, pears, peaches, apricots, walnuts, and other fruit trees; orchids, carnations, roses, and other flowers; cacao; poplar, elms, and other deciduous trees; pine, Douglas-fir, spruce, and other conifers; turf grasses; cacao; and rubber trees and other members of the genus Hevea.
In yet another embodiment, the invention provides methods for expressing proteins in host cells, such as plant host cells. Such methods involve operably linking a promoter, such as those described above, to at least one ORF to produce a transgene, and introducing the transgene into a plant. Accordingly, the invention also provides proteins that are produced by these methods.
The PmBiP promoter (SEQ ID NO: 31) is shown herein to be inducible (at least via wounding and probably via cold temperatures), and the amount of mRNA encoding PmBiP protein as well as BiP protein itself has been shown to be increased at cold temperatures, thus making the PmBiP promoter ideal for use in the expression of proteins. This is because cold temperatures serve to stabilize the protein during translation. Accordingly, another aspect of the invention provides inducible promoters derived through the use of fragments of the promoter described herein.
An alternative method of characterizing promoters is by analyzing the various promoter elements found within a promoter sequence. Hence, the invention also provides promoters that maintain promoter activity and include at least 8 promoter elements selected from the group consisting of the E-box motif (SEQ ID NO: 1), the MNF1 element (SEQ ID NO: 28), the POLLEN1LELAT52 element (SEQ ID NO: 29), the ROOTMOTIF element (SEQ ID NO: 30), the 2SSEEDPROTBANAP element (SEQ ID NO: 32), the BOXIIPCCHS element (SEQ ID NO: 33), the ASF1 MOTIF element (SEQ ID NO: 34), ACGT-core elements (SEQ ID NO: 4), the CAAT-box (SEQ ID NO: 9), the CANABNNAPA element (SEQ ID NO: 12), the HEXMOTIF element (SEQ ID NO: 27), and duplicates thereof, wherein the promoter displays promoter activity. The invention also provides promoters that contain the following promoter elements in the following orientation: 3xe2x80x2-ACGT-core element (SEQ ID NO: 4), E-box motif (SEQ ID NO: 1), CAAT-box (SEQ ID NO: 9), 2SSEEDPROTBANAP (SEQ ID NO: 32), CANABNNAPA element (SEQ ID NO: 12), HEXMOTIF element (SEQ ID NO: 27), CAAT-box (SEQ ID NO: 9), BOXIIPCCHS element (SEQ ID NO: 33), E-box motif (SEQ ID NO: 1), ASF1MOTIF (SEQ ID NO: 34), POLLEN1LELAT52 element (SEQ ID NO: 29), and MNF1 element (SEQ ID NO: 28) -5xe2x80x2.
Finally, the invention also provides vectors, host cells, and transgenic plants that include the promoters described above by their inclusion of various promoter elements.
These and other aspects of the invention will become readily apparent from the following detailed description.